Protein recognition of specific DNA sequences is critical for the proper regulation of transcription. Binding specificity is accomplished through a series of direct and solvent-mediated protein-DNA contacts. The intrinsic sequence-dependent ability of DNA to assume specific conformations can also play a role in specificity. Small molecule effectors, acting as an environmental signals, may bind to the regulator to induce needed conformational changes for specific DNA binding. Recognizing such signals is crucial for precise transcription regulation and requires effector binding to display high affinity. Improper spatial or temporal transcription can lead to uncontrolled cellular proliferation or cell death. Thus, the complete elucidation of the mechanisms by which environmental signals are transduced into the biological response of transcription repression requires the combination of crystallographic, biochemical and thermodynamic studies. As a model system, we are continuing our structural and biochemical studies on the E. coli Purine Repressor (PurR), and allosterically regulated transcription repressor. The five specific aims are to: (1) Extend the resolution of the PurR-hypoxanthine/guanine-purF (and purF (pal) operator ternary complexes from 2.7 cross of the circle to at least 2.2. cross of the circle resolution and the corepressor-free CBD to at lest 2.0 cross of the circle resolution. (2) Determine the structures, binding affinities and thermodynamic properties of PurR-corepressor-purF operator complexes in which PurR residues involved in major groove binding, Thr15, Thr16, His20 and Arg20, or minor groove binding, Val50, Ala51, Ser53, Leu54, Lys55 and Val56, are substituted. (3) Determine the structures and DNA and corepressor binding affinities of PurR-corepressor-purF operator complexes in which PurR residues Arg190, Thr192 and Glu222, which line the corepressor binding pocket, are substituted. (4) Determine the structures and DNA and corepressor binding affinities of PurR-corepressor-ppurF operator complexes and the structures of the unliganded Corepressor Binding Domain (residues 53-341) of PurR in which residues Tyr 73, Phe74, Tyr107, Trp147, Asp160, Asn161 and Gln292 are substituted. (5) Crystallize and determine the structures and binding affinities of PurR- hypoxanthine-DNA operator complexes (a) in which purF operator base pair A8:T8' has been replaced by a T:A, C:G or G:C base pair or (b) in which the purF central C9pG9' base pair step is replaced with CpI, TpDAP, TpAP, dUpDAP, dUpAP, GpC and ApT and TpA steps.